Fig 1.
Temporal dynamics of the meiotic division program and its control by the SAC.
(A) Progression through the meiotic divisions was analyzed by time lapse imaging with spermatocyte cysts expressing His2Av-mRFP and Cid/Cenp-A-EGFP. Representative still frames with a single spermatocyte illustrate the different division phases. Time (hours:minutes:seconds) is given with zero corresponding to start of the movie where spermatocytes were at the S6 stage. Only one daughter cell is shown in telophase. Scale bar = 5 μm. (B) Dot plots representing the duration of the distinct division phases during M I and M II in different cysts (n ≥ 9 cysts, each from a different testis) as well as the average durations (+/- s.d.). (C) Loss of SAC function accelerates anaphase onset. During M I and M II, the duration of entry (NEBD until anaphase onset) and exit (anaphase onset until interphase onset) was determined in control and mad2 null mutants. Averages +/- s.d. are from n ≥ 29 cells from at least seven different testes.
Fig 2.
Spindle assembly and MT interaction with KTs.
Time lapse imaging was performed with spermatocytes expressing GFP-βTub56D (GFP-betaTub) and tdTomato-Cenp-C (tdTom-CenpC) (A) Maximum intensity projections of optical sections (3–4 with 300 nm spacing) containing the pole to pole axis at the indicated times (minutes:seconds after NEBD I) are displayed. Arrows indicate initial formation of intranuclear MTs that are eventually bundled into the poles of the intranuclear spindle (arrowheads). (B) All KTs of a spermatocyte were tracked during M I (NEBD until end of metaphase) and their velocity (VKT) was plotted. The four bivalents were labeled with a capital letters (chr A-D) and the two KTs of a bivalent with small letters (a and b). The prominent KT jumps (j1-3) are further documented in panels C and D. (C) KT tracks representing the prominent jumps of KT pairs (j1-3) indicated in panel B. The early jumps (j1 and j2) are displayed in the top panel and a late jump (j3) in the bottom panel. In both panels, a still frame from the time point just after the jump is combined with KT tracks covering the preceding period of 15 time points (150 seconds). Tracks are displayed with a color code reflecting VKT from slow (blue) to fast (red). The KTs are indicated by spheres with pairs displayed in the same color as also used in the VKT curves (B). (D) GFP-βTub56D signals in the slices containing the jumping KTs (j1-3) are displayed at three consecutive time points (minutes:seconds after start of imaging) with maximum intensity projections (2–3 optical sections with 300 nm spacing). Arrows indicate KT positions prior to and arrowheads those after the KT jump. Scale bars = 5 μm.
Fig 3.
Progression through M I in the absence of KT function.
(A-D) The kinetochore protein Spc105 was depleted in spermatocytes expressing Cid-EGFP and His2Av-mRFP. After time lapse imaging, all eight KTs in a representative spermatocyte were tracked during M I. (A) The KT tracks during consecutive division phases (time in minutes:seconds) are displayed as an overlay on still frames from the end of the corresponding phases. The two KTs associated with the same bivalent are represented by colored spheres with red, yellow, blue and green indicating chr XY, 2, 3 and 4, respectively. Transport of bivalents into the central region followed by aberrant segregation of unseparated KT pairs to opposite poles along horizontal axis is apparent. Scale bar = 3 μm. (B) KT velocities (VKT) plotted over time. (C) Angles (AKT) between the spindle axis and the axis connecting the two KTs of a bivalent plotted over time. (D) Distances (DKT) between the two KTs of a bivalent plotted over time. (E) Spc105 is required for recruitment of Mis12 to the KT. Spc105 was depleted in spermatocytes expressing Mis12-EGFP and His2Av-mRFP. Still frames after live imaging document the time point in prometaphase I where Mis12-EGFP signals at KTs are maximal in controls. (F) Spc105 is required for recruitment of Bub3 to the KT and for SAC function. Spc105 was depleted in spermatocytes expressing EGFP-Bub3 and His2Av-mRFP. Still frames after live imaging document the time point in prometaphase I where EGFP-Bub3 signals at KTs are maximal in controls. Dot plots indicate the duration of M I and M II as well as averages (+/- s.d., n ≥ 10 cells from at least six different cysts) in spermatocytes without (control) and with Spc105 depletion (Spc105 RNAi). (G) Cid-EGFP dot counts per nucleus in daughter cells generated by M I and M II, respectively. Four dots per nucleus are observed in normal cells (control) and variable numbers after Spc105 depletion (Spc105 RNAi). (n) number of analyzed cells. (H) Chromosome transport into the central region is evident after Spc105 depletion (Spc105 RNAi) but not in the presence of colcemid. Representative still frames at NEBD I (00:00) and 15 minutes later are shown with dotted yellow circles indicating the region occupied by chromosomes. Scale bars = 5 μm.
Fig 4.
Efficiency of chromosome bi-orientation during unperturbed M I revealed by KT tracking.
(A-D) All KTs of a representative spermatocyte expressing Mis12-EGFP and His2Av-mRFP were tracked during M I and assigned to chromosomes. (A) The KT tracks observed during the indicated phases are displayed as an overlay on the still frames from the end of the corresponding phases. Tracks are displayed with a color code reflecting VKT from slow (blue) to fast (red). (B) VKT plotted over time. (C) AKT plotted over time. (D) DKT plotted over time. Black dotted lines indicate division phase transitions. (E) Average DKT at late metaphase I (+/- s.d., n = 6 cells). DKT in case of the small chromosome 4 is less than in other bivalents (XY, chr 2 and chr 3). (F, G) Two distinct events of re-orientation of a KT pair during M I are illustrated with still frames oriented so that the spindle axis is horizontal. The KT tracks over the preceding two time points are superimposed on the images. Yellow arrows indicate the direction of movement of the re-orienting KT, which is to the right in the top panel and to the left after re-orientation in the bottom panel. The time interval separating the two still frames is indicated (minutes:seconds) Scale bars = 2 μm.
Fig 5.
Behavior of KTs of univalents in mnm mutants during M I.
After time lapse imaging with spermatocyte cysts from mnm mutant flies expressing Cid-EGFP and His2Av-mRFP, all eight KTs were tracked and assigned to chromosomes. (A) VKT values plotted over time from a representative spermatocyte. Onset of the indicated phases marked by dotted lines. Metaphase onset was scored when the rapid KT jumps vanished rather than after bi-orientation of the last bivalent as in controls. (B) Comparison of KT tracks in control (left) and mnm mutants (right). The track of one representative KT from NEBD until metaphase onset is displayed as an overlay on the still frame from metaphase onset. Track color corresponds to time from early (blue) to late (red). White dotted lines demarcate the metaphase plate region (width 3 μm corresponding to about ¼ of the pole-to-pole distance). Scale bar = 3 μm. (C) State of sister KT conjunction during M I after Spc105 depletion in spermatocytes with and without mnm and snm function. The normal number of eight Cid-EGFP dots was detected by time lapse imaging with spermatocytes from the indicated genotypes. Still frames from representative spermatocytes during early prometaphase I are displayed. Scale bar = 5 μm.
Fig 6.
Sister KTs separation and bi-orientation during M II.
(A) Sister KT behavior during progression through the meiotic divisions as observed by time lapse imaging with spermatocytes expressing Cid-EGFP and His2Av-mRFP is illustrated by representative still frames displaying the Cid-EGFP signal associated with one pair of sister KTs (except for telophase II where a single sister KT is shown). Sister KTs are detected as a single Cid-EGFP dots (yellow circle) from NEBD I to NEBD II until unequivocal separation in metaphase II (yellow arrows). (B) Time lapse imaging revealed occasional transient splitting of Cid-EGFP dots (white arrows), as illustrated with an event during IK. Time (seconds) is indicated. Scale bar = 2 μm. The scatter plot describes separation width and duration of such events in the indicated genotypes (n = 13 in controls, n = 36 after Spc105 RNAi). (C-E) KTs in spermatocytes expressing His2Av-mRFP and Mis12-EGFP were tracked during M II followed by plotting of VKT (C), AKT (D) and DKT (E) over time. Black dotted lines mark the onset of the indicated division phases. (F) Spermatocytes expressing Mis12-EGFP and His2Av-mRFP were used for time lapse imaging of M II in the absence (control) and presence of colcemid. Inter sister KT distances (DKT) were measured at the indicated time points (minutes after NEBD II) and plotted with averages (+/- s.d.). (G) Separation of a sister KT pair during prometaphase II is documented with high magnification views (top) and a graph (bottom) of the distance between the two sister KTs (yellow line in stills) at the indicated time points (seconds after NEBD II). A track overlay in the top panel describes the displacement of one sister KT from the preceding to the displayed time point. Track color indicates VKT from slow (blue) to fast (red). Spindle axis is vertical. After 620 sec, the sister KT pair remained stably bi-oriented during congression into the metaphase plate (located at the upper edge of the images) until anaphase. Scale bar = 0.7 μm.
Fig 7.
Bi-orientation of univalents and Fzy/Cdc20-dependent separation of their sister KTs during M I.
For an analysis of the behavior of sister KTs associated with univalents during M I, time lapse imaging was performed with mnm (A,B) and tef (C,D) mutant spermatocytes expressing Cid-EGFP and His2Av-mRFP. (A) Univalents were assigned to three classes according to position and shape of the associated Cid-EGFP signals during exit from M I (left panel). Class I univalents have sister KTs within the metaphase plate and stretched apart, indicating bi-orientation. In class II univalents, sister KTs are also within the metaphase plate but unresolved, and class III univalents have unresolved sister KTs close to a spindle pole. High magnification views (right panel) display the Cid-EGFP signals of the indicated univalents (I, II, III) at different time points (t1, t2, t3; see also B). Scale bar = 3 μm (left panel) and 0.5 μm (right panel). (B) Distance between the two sister KTs of the indicated univalents plotted over time. The dotted lines mark the indicated division phase transitions. The arrows (t1, t2, t3) mark the time points displayed in panel A on the right. (C) Still frames from different time points (see also D). The KTs of the XY bivalent (white arrows) and of representative univalents (colored arrowheads) are marked. Scale bar = 2μm. (D) Distance between the two sister KTs of the univalents indicated in (C) plotted over time (y axis on the left), as well as separation of the KTs on the X and Y chromosome (DKT XY) (y axis on the right). The rapid increase in DKT XY reveals anaphase onset. The arrows (t1-t4) mark the time points displayed in (C). (E) The overall duration of M I was determined in spermatocytes with the indicated genotypes. Averages (+/- s.d.) are indicated in red. n > 10 cells from at least 3 different testes.